Mass spectrometry method for measuring thiamine in body fluid

ABSTRACT

Provided are methods for determining the amount of total thiamine in a body fluid sample using liquid chromatography and mass spectrometry. Total thiamine is converted to free thiamine by treatment with an acid phosphatase prior to thiamine separation and quantification.

FIELD OF THE INVENTION

The invention relates to the detection of vitamin B1 (thiamine).

BACKGROUND OF THE INVENTION

Thiamine, also known as vitamin B1, is a water-soluble vitamin thatexists in the body as free thiamine and in one of several phosphorylatedforms including thiamine monophosphate (ThMP), thiamine pyrophosphate(ThPP), thiamine triphosphate (ThTP), and adenosine thiaminetriphosphate (AThTP). In human body, the primary vitamin B1 in serum andplasma is free thiamine while ThPP predominates in cellular components,for example blood cells. ThMP and ThTP only exist in very limitedamounts (under detection level) in body fluid. The AThTP was recentlydiscovered in Escherichia coli (Nature Chemical Biology 3, 211-212,2007). Its existence and significance in human have not been reported atpresent.

Thiamine and its derivatives play an important role in the metabolism oflipids and carbohydrates. ThPP, also known as thiamine diphosphate(ThDP), is the major biologically active form of thiamine and is arequired coenzyme for a number of enzymes involved in carbohydratemetabolism and nervous system function including the biosynthesis oflipids and acetylcholine. Thiamine-requiring enzymes include, forexample, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase,branched-chain α-ketoacid dehydrogenase, transketolase, and2-hydroxyphytaoyl-CoA lyase.

Thiamine in food is easily decomposed by heat and particularly underalkaline conditions. After oral ingestion, thiamine is readily absorbedby both active transportation at low concentrations (<5 mg/dL) andpassive diffusion at higher concentration levels. Phosphorylation in thejejunal mucosa produces ThPP.

Thiamine deficiency is frequently caused either by an inadequate intakeof thiamine-rich foods (e.g., peas, spinach, liver, beef, and bananas),impaired absorption (genetic), over consumption of thiaminase-rich foods(e.g., raw fish and shellfish) or food high in anti-thiamine factors(e.g., tea and coffee), general malnutrition, or alcoholism. Thiaminedeficiency results in a variety of diseases with a myriad of symptomsaffecting virtually every system in the body. Beriberi, for example, isa condition induced by thiamine deficiency which is characterized byperipheral neuropathy resulting in abnormal reflexes, diminishedsensation, muscle pain and weakness, and seizures. “Wet” beriberiaffects the cardiovascular system, with peripheral edema and tachycardiadue to congestive heart failure. The cerebral form of the disease mayresult in Wernicke's encephalopathy, Korsakoff's psychosis, orWernicke-Korsakoff Syndrome.

SUMMARY OF THE INVENTION

The present invention relates to the detection and quantification oftotal thiamine in a body fluid sample. Phosphorylated thiamine in thebody fluid is converted to free thiamine by hydrolysis using aphosphatase. The free thiamine is recovered by an organic solventextraction and purified by liquid chromatography. Mass spectrometry (MS)is used to quantify the amount of free thiamine, which is related to theconcentration of total thiamine present in the body fluid sample.

Accordingly, the invention provides a method for determining the amountof total thiamine in a body fluid sample by (i) removing soluble proteinfrom the sample, (ii) treating the sample with an acid phosphatase toconvert phosphorylated thiamine into free thiamine; (iii) performing anorganic solvent extraction on the sample following the acid phosphatasetreatment of step (ii); (iv) purifying the free thiamine by liquidchromatography from the aqueous phase that results from the organicsolvent extraction of step (iii); and (v) determining the amount of freethiamine by mass spectrometry, wherein the amount free thiamine isrelated to the amount of total thiamine in the body fluid sample, andthe amount of total thiamine in the body fluid sample is determined.

In preferred embodiments, soluble protein is removed in step (i) usingacid precipitation. Suitable acids include, for example, perchloricacid, hydrochloric acid, sulfuric acid, nitric acid, boric acid, andacetic acid. Most preferably, soluble protein is removed using 7%perchloric acid. Following precipitation, the acid insoluble protein maybe removed by any suitable method including, for example, filtration orcentrifugation.

In preferred embodiments, a body fluid sample is obtained from a mammal,preferably a primate (e.g., a human), a dog, a cat, or a horse. Suitablebody fluids include whole blood, plasma, serum, urine, saliva, tears,and cerebrospinal fluid.

In preferred embodiments, an phosphatase is used under acidic condition(pH 4.6±0.1) and the phosphatase reaction converts substantially all(e.g., at least 80%, 85%, 90%, 95%, 99%, or 100%) of the phosphorylatedthiamine into free thiamine. In other embodiments, the phosphatase is anacid phosphatase including, for example, thiamine pyrophosphatase.

The organic solvent extraction comprises mixing an organic solvent withthe aqueous bodily fluid for a sufficient time that lipophilicimpurities dissolve in the organic solvent and the phosphatase reactionis terminated. In preferred embodiments of the organic solventextraction, the organic solvent is tetrahydrofuran, benzene, toluene,diethyl ether, chloroform, ethyl acetate, or dichloromethane. Mostpreferably, the organic solvent is chloroform.

In one embodiment, the liquid chromatography is high performance liquidchromatography (HPLC). Preferably, the HPLC is reverse phase HPLC. Morepreferably, the HPLC utilizes a C18 analytical chromatography column. Inanother preferred embodiment, the HPLC utilizes a mobile phase gradient(e.g., a step gradient or a continuous gradient) comprising an aceticacid buffer and acetonitrile.

In one preferred embodiment, the thiamine-containing effluent of theHPLC column is directed, in an in-line format, into a mass spectrometry(MS) device where it is ionized to a parent ion in the MS device. Oneuseful method of ionization is electrospray ionization. Preferably, thethiamine parent ion has a mass/charge ratio of 265.00±1.00. In oneembodiment, the thiamine parent ion quantified by the MS operating inpositive ion mode. In another embodiment, the thiamine parent ion isfurther isolated using a first MS quadrupole analyzer, followed byfragmentation into one or more thiamine daughter ions. One or morethiamine daughter ions are subsequently detected and/or quantified bythe MS operating in positive ion mode. Preferably, the one or morethiamine daughter ions include an ion having a mass/charge ratio of144.00±1.00 or 121.94±1.00. In one embodiment, the thiamine daughterions are formed by collision induced dissociation using an inert gas.Preferable inert gases include, for example, argon, helium, andnitrogen.

In one embodiment, a separately detectable internal standard is added tothe body fluid sample. Preferably, the internal standard is added to thebody fluid prior to the first processing step. Preferred internalstandards include, for example, pyrithiamine and isotopically labeled(e.g., deuterated, or carbon-13 labeled) thiamine or the correspondingsalt forms. For methods in which pyrithiamine is used as an internalstandard, preferably the pyrithiamine is ionized to parent ion having amass/charge ratio (m/z) of 259.04±1.0 prior to the first MS detection orisolation. It is also preferable that the pyrithiamine parent ion isfragmented to a daughter ion having a mass/charge ratio of 122.00±1.0m/z for detection and quantification by the second MS.

The term “operating in positive ion mode” refers to those massspectrometry methods where positive ions are detected. Similarly,“operating in negative ion mode” refers to those mass spectrometrymethods where negative ions are detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatogram showing the elution of 15 nmol/L thiamine froman EDTA-plasma sample using the sample preparation and HPLC proceduresdescribed herein. Thiamine elutes with a peak retention time of about0.44 minutes.

FIG. 2 is a chromatogram showing the elution of the internal standardpyrithiamine) from a spiked EDTA-plasma sample using the samplepreparation and HPLC procedures described herein. The internal standardelutes with a peak retention time of about 0.44 minutes.

FIG. 3 is a graph showing a typical standard curve of thiamine purifiedand quantified using the LC/MS/MS method provided herein. The standardcurve plots the ratio of the area of the thiamine peak to the area ofthe IS peak (“peak area ratio”) versus the known concentration ofthiamine in the calibration standards. Linear regression was performedon the data and the calculated regression line (r²=0.9998) is shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for determining the amount oftotal thiamine in a sample of body fluid (e.g., serum, plasma, and wholeblood). The method first removes soluble protein (e.g., by acidprecipitation), followed by an acid phosphatase treatment which convertsphosphorylated thiamine into thiamine. The acid phosphatase reaction isterminated by an organic solvent (e.g., chloroform) extraction and thethiamine is purified using liquid chromatography; preferably highperformance liquid chromatography. Thiamine is quantified using massspectrometry. In preferred embodiments, the thiamine is quantified bytandem mass spectrometry (MS/MS) in which the thiamine is first ionizedto a parent ion which is isolated by a first mass spectrometer. Theisolated parent ion is then fragmented into one or more characteristicdaughter ions and quantified by a second mass spectrometer.

The inventive method may be adapted to a high-throughput format. Theassay offers enhanced sensitivity, specificity, and is accomplished inless time and with less sample preparation than required by otherthiamine assays. In various embodiments the methods of the inventionaccurately quantify total thiamine concentrations as low as 6 nmol/L.Typically, however, total thiamine detected in plasma and serum is about8-30 nmol/L and 78-185 nmol/L in whole blood.

The term “total thiamine” as used herein refers to all forms of thiaminepresent in a sample of body fluid. Specifically included in “totalthiamine” are free thiamine, and thiamine converted from thiaminemonophosphate (ThMP), thiamine pyrophosphate (ThPP), and thiaminetriphosphate (ThTP).

The term “analytical column” as used herein refers to a chromatographycolumn having sufficient chromatographic “plates” to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such a columnis often distinguished from an “extraction column,” which has thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. A preferred analytical column is an HPLC column.

Sample Collection

The thiamine concentration may be assessed in any body fluid. Mostconveniently, thiamine is assessed in whole blood or a blood fractionsuch as plasma, serum. Standard techniques for obtaining, processing,storing, and shipping these thiamine-containing body fluids may be used.It is well-known that thiamine is light-, and heat-sensitive so careshould be taken throughout the sample collection and assay procedure toprotect the sample against light and heat exposure that would result insignificant thiamine degradation.

In preferred embodiments, plasma and serum samples are processed fromwhole blood within four hours of receipt by the laboratory. Desirably,the samples are maintained at about −10° C. to about −30° C. untilassayed for thiamine. It is also preferred if the subject has fasted forabout 12 hours prior to blood sampling. Further, it is desirable thatthe subject refrains for 24 hours prior to blood sampling from consumingalcohol, coffee, tea, raw fish, raw shell fish, and vitamin supplementscontaining thiamine.

Protein Removal

Prior to performing the acid phosphatase reaction, it is desirable toremove potentially interfering molecules. One such processing stepincludes performing a protein removal step. The removal of potentiallyinterfering proteins may be accomplished by any appropriate methodincluding, for example, liquid chromatography, acid precipitation,filtration, centrifugation, and the like. Protein removal by filtrationtypically employs either spin filters or pressure filters having a poresize of ≦0.22 μm.

In a preferred embodiment, protein is removed from the sample byperforming a protein precipitation reaction. This is achieved by addinga precipitating agent (e.g., an acid) to the body fluid sample, mixingthe agent and the sample for a time and under conditions sufficient tocause protein precipitation, and then separating the precipitatedproteins from the thiamine-containing solution. In a preferredembodiment, an equal amount of 7% aqueous perchloric acid is added tothe body fluid sample, followed by vigorous mixing and incubation at 15°C. for about 5-30 minutes. The samples are centrifuges to pellet theprotein and other acid-insoluble matter (e.g., 3000 rpm for 10 minutesat 2-8° C.), and the thiamine-containing supernatant is removed forfurther processing.

Acid Phosphatase Reaction

The present invention provides a method for detecting total thiamine ina single assay. The inventive method converts phosphorylated thiamine(e.g., ThMP, ThPP, and ThTP) into free thiamine in order that only asingle thiamine species is detected and quantified. Conversion ofphosphorylated thiamine into free thiamine may be done by any convenientmethod including, for example, treatment of the thiamine-containingsample with an acid phosphatase. Suitable acid phosphatases include, forexample, those derived from plant sources (e.g., potato, sweet potato,and wheat germ), and mammalian sources (e.g., human and bovine) which isused at about 1-30 mg/ml, preferably about 10 mg/ml. It is desirablethat the acid phosphatase reaction is run to substantial completion(i.e., that substantially all of the phosphorylated thiamine ishydrolyzed to free thiamine). Typically, the acid phosphatase reactionis performed under acidic condition (pH 4.6±0.1) at about 40° C. and isrun for about one to two hours for serum and plasma samples or 12+ hours(e.g., overnight) for whole blood samples.

Organic Solvent Extraction

Following the acid phosphatase hydrolysis reaction, the body fluidsample contains a variety of salts, proteins (including the acidphosphatase enzyme), and other impurities, which desirably should beremoved prior to mass spectrometry. Organic solvent extraction is auseful method to both terminate the hydrolysis reaction and removelipophilic impurities from the thiamine-containing sample.

An organic solvent (e.g., chloroform) is added to the acid phosphatasereaction vessel in sufficient quantity and for a sufficient duration toterminate the enzymatic reaction. Typically, about 1-20 volumes (e.g., 5volumes) of organic solvent is added, the solution is vigorously mixed,and the organic and aqueous phases are separated. Phase separation maybe expedited by mild centrifugation (e.g., about 3000 rpm for about 10minutes). The thiamine-containing aqueous phase is recovered for furtherprocessing and the organic phase is discarded.

Thiamine Separation by Liquid Chromatography

Following organic solvent extraction, the thiamine is further purifiedfrom the aqueous phase by liquid chromatography (LC) prior toquantification using mass spectrometry. Liquid chromatography removesaqueous impurities and may be used to concentrate the thiamine fordetection. Traditional LC relies on chemical interactions between samplecomponents (e.g., thiamine) and a stationary phase such as a columnpacking. Laminar flow of the sample, mixed with a mobile phase, throughthe column is the basis for separation of the components of interest.The skilled artisan understands that separation in such columns is apartition process.

In one embodiment, the thiamine is separated using high pressure liquidchromatography (HPLC). The skilled artisan recognizes a variety ofsuitable HPLC columns, mobile phases, and HPLC conditions suitable forthiamine separation. Preferably, the HPLC is reverse phase HPLC and/orthe HPLC column is a C18 analytical column. The skilled artisan alsorecognizes a variety of mobile phases and mobile phase combinationsuseful for reverse phase HPLC. Preferably, the elution from the HPLCcolumn is driven by a binary step-gradient of aqueous ammonium acetate(10 mM) and acetonitrile (100%).

In various embodiments, one or more of the purification and/or analysissteps can be performed in an automated fashion. By careful selection ofvalves and connector plumbing, two or more chromatography columns can beconnected as needed such that material is passed from one to the nextwithout the need for any manual steps. In preferred embodiments, theselection of valves and plumbing is controlled by a computerpre-programmed to perform the necessary steps. Most preferably, thechromatography system is also connected in-line to the detector system,e.g., an MS system. Thus, an operator may place a tray of hydrolyzed andpurified samples in an autosampler, and the remaining operations areperformed under computer control, resulting in purification and analysisof all samples selected. In one embodiment, a diverter valve is placedin-line between the LC column and the interface with the MS. Thediverter valve directs the LC effluent into a waste container untilslightly prior to the time expected thiamine peak retention (e.g., peakretention time minus 0.1, 0.15, 0.2, or 0.25 minutes). This prevents thehigh salt solvent front and other impurities from being passed into theMS device.

As used here, “in-line” refers to a configuration in which the LC andthe ionization/injection device for the first MS quadropole arefunctionally connected in order that the LC effluent passes directlyinto the first MS device. “In-line” configurations may include aselector valve such that the effluent from two or more LC columns may bedirected individually into the MS device and, optionally, to a wastecontainer. Such a configuration is useful for a high throughput systemand reduces the analysis time required for a large number of samples.High throughput systems may be designed in which an autosamplerinitiates LC purifications on the two or more LC columns at staggeredintervals. In this way, the purified thiamine peak is eluted from eachLC column at a known interval. Preferably, the purified thiamine peakseluting from the two or more LC columns are directed into the MS devicein rapid succession, but with sufficient temporal separation thatindividual measurements are not compromised. Such a high throughputsystem reduces the amount of “idle-time” for MS detection attributableto the LC procedure which typically requires more time than the MSanalysis.

By contrast, “off-line” refers to a configuration that requires manualintervention to transfer the LC effluent to the MS device. Typically,the LC effluent is captured by a fractionator and must be manuallyloaded into a MS device or into an autosampler for subsequent MSdetection. Off-line configurations are useful, but less desirablebecause of the increased time required to process large numbers ofsamples.

Thiamine Analysis by Mass Spectrometry

Thiamine purified by LC is conveniently detected and quantified by massspectrometry (MS). The thiamine-containing effluent from the LC isinjected into an ionization chamber of the MS in which a first (parent)ion is produced. The parent ion may be detected directly in a first MS,or it may be isolated by the first MS, fragmented into characteristicdaughter ions, and one or more of the daughter ions detected in a secondMS (i.e., tandem MS).

The term “mass spectrometry” or “MS” as used herein refer to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “m/z.” In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”).

As used herein, the term “ionization” refers to the process ofgenerating an analyte ion having a net electrical charge equal to one ormore electron units. Negative ions are those having a net negativecharge of one or more electron units, while positive ions are thosehaving a net positive charge of one or more electron units.

The ions may be detected using several detection modes. For example,selected ions may be detected using a selective ion monitoring mode(SIM) which includes multiple reaction monitoring (MRM) or selectedreaction monitoring (SRM). Alternatively, ions may be detected using ascanning mode.

Preferably, the mass-to-charge ratio is determined using a quadrupoleanalyzer. For example, in a “quadrupole” or “quadrupole ion trap”instrument, ions in an oscillating radio frequency field experience aforce proportional to the DC potential applied between electrodes, theamplitude of the RF signal, and m/z. The voltage and amplitude can beselected so that only ions having a particular m/z travel the length ofthe quadrupole, while all other ions are deflected. Thus, quadrupoleinstruments can act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

“Tandem mass spectrometry,” or “MS/MS” is employed to enhance theresolution of the MS technique. In tandem mass spectrometry, a parention generated from a molecule of interest may be filtered in an MSinstrument, and the parent ion subsequently fragmented to yield one ormore daughter ions that are then analyzed (detected and/or quantified)in a second MS procedure.

Collision-induced dissociation (“CID”) is often used to generate thedaughter ions for further detection. In CID, parent ions gain energythrough collisions with an inert gas, such as argon, and subsequentlyfragmented by a process referred to as “unimolecular decomposition.”Sufficient energy must be deposited in the parent ion so that certainbonds within the ion can be broken due to increased vibrational energy.

By careful selection of parent ions using the first MS procedure, onlyions produced by certain analytes of interest are passed to thefragmentation chamber to generate the daughter ions. Because both theparent and daughter ions are produced in a reproducible fashion under agiven set of ionization/fragmentation conditions, the MS/MS techniquecan provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation can be used to eliminateinterfering substances, and can be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each m/z over a given range (e.g., 10 to1200 amu). The results of an analyte assay, that is, a mass spectrum,can be related to the amount of the analyte in the original sample bynumerous methods known in the art. For example, given that sampling andanalysis parameters are carefully controlled, the relative abundance ofa given ion can be compared to a table that converts that relativeabundance to an absolute amount of the original molecule. Alternatively,molecular standards (e.g., internal standards and external standards)can be run with the samples, and a standard curve constructed based onions generated from those standards. Using such a standard curve, therelative abundance of a given ion can be converted into an absoluteamount of the original molecule. In certain preferred embodiments, aninternal standard is used to generate a standard curve for calculatingthe quantity of thiamine. Numerous other methods for relating thepresence or amount of an ion to the presence or amount of the originalmolecule are well known to those of ordinary skill in the art.

The skilled artisan will understand that the choice of ionization methodcan be determined based on the analyte to be measured, type of sample,the type of detector, the choice of positive versus negative mode, etc.Ions can be produced using a variety of methods including, but notlimited to, electron ionization, chemical ionization, fast atombombardment, field desorption, and matrix-assisted laser desorptionionization (MALDI), surface enhanced laser desorption ionization(SELDI), photon ionization, electrospray ionization, and inductivelycoupled plasma. Electrospray ionization is a preferred ionizationmethod. The term “electrospray ionization,” or “ESI,” as used hereinrefers to methods in which a solution is passed along a short length ofcapillary tube, to the end of which is applied a high positive ornegative electric potential. Solution reaching the end of the tube, isvaporized (nebulized) into a jet or spray of very small droplets ofsolution in solvent vapor. This mist of droplets flows through anevaporation chamber which is heated to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

Desirably, the effluent of the LC is injected directly and automatically(i.e., “in-line”) into the electrospray device. In preferredembodiments, the thiamine contained in the LC effluent is first ionizedby electrospray into a parent ion of about 265.00±1.00 m/z. The firstquadropole of the MS/MS is tuned to be a mass filter for the thiamineparent ion (and/or the internal standard).

Parent ion(s) passing the first quadropole are then ionized and/orfragmented prior to passing into the second quadropole. In preferredembodiments, the ions are collided with a inert gas molecule in aprocess of collision-induced dissociation (CID). Suitable inert gasesinclude, for example, argon, helium, nitrogen, etc. Desirably, thethiamine parent ion is fragmented into daughter ions havingm/z=144.03±1.00 and/or m/z=121.94±1.00. It is these daughter ions thatare subsequently detected.

Standards

It is desirable to use one or more standards for calibration andquantification purposes. Internal and external standards are commonlyused for these purposes. Internal standards are typically analogs of thecompound(s) of interest that are expected to react similarly during allextraction and quantification steps. A known amount of an internalstandard is typically added to each sample early in the processing inorder to account for any loss of compound during any processing step.External standards are typically consist of samples containing a knownquantity of the compound of interest, or an analog, which are processedin parallel with the experimental samples. External standards are oftenused to control for the efficiency of the various processing steps.Finally, calibration standards are used to quantify the amount of thecompound of interest in each experimental and external control sample.Typically, a series of calibration standards containing varying knownamounts of the compound(s) of interest are injected directly into thedetection device (i.e., the MS). Calibration standards are used togenerate a standard curve, against which the experimental samples arequantified. These standards also may be used to determine limits ofdetection for any particular detection methodology.

Internal Standards

Typically the internal standard is added in a known quantity to thesample of body fluid immediately after thawing and prior to the firstextraction or purification step. A suitable internal standard that maybe used is pyrithiamine (e.g., pyrithiamine hydrobromide; Sigma-Aldrich,Catalog No. P0256). Pyrithiamine, when subjected to the LC-MS-MSconditions described herein gives rise to a parent ion havingm/z=259.04±1.00 and a daughter ion having m/z=122.00±1.00.Alternatively, deuterated or carbon-13 labeled thiamine may be used asan internal standard.

External Standards

Thiamine or any of its phosphorylated forms (e.g., ThMP, ThPP, ThTP, andAThTP) may be used as an external standard. Typically, the externalstandard is prepared in a medium that closely resembles the medium ofthe experimental samples and is processed in parallel with theexperimental samples. The phosphorylated forms of thiamine are preferredas external standards in order to control for the efficiency of the acidphosphatase reaction. One useful medium is analyte-stripped anddelipidized human serum (Biocell™; Biocell Labs, Carson, Calif.; catalogno. 1131-00).

Calibration Standards

Thiamine, the compound of interest, is preferably used as thecalibration standard in order to create a calibration curve. Generally,the calibration curve covers thiamine concentrations from about thelimit of detection to a concentration at least two-fold greater(preferably at least an order of magnitude greater) than the highestexpected thiamine value in an experimental sample. When an internalstandard is used in the experimental samples, it is desirable to add asimilar amount of the internal standard to the calibration standards andto create a standard curve based on the ratio of the thiamine signal(e.g., peak area) to internal standard signal. This is referred to asthe area ratio is useful to eliminate some inter-sample variability thatmay be present for calibration curves based solely on the thiamineconcentration.

Blanks

Blanks are samples processed in parallel with the experimental samplesand/or positive controls which contain a similar (or the same) medium asused to prepare the positive controls and,or is similar to the bodyfluid in which thiamine is being measured. One preferred medium usefulas both a blank and as the medium for the positive controls isanalyte-stripped and delipidized human serum (Biocell™; Biocell Labs,Carson, Calif.; catalog no. 1131-00). Another useful medium isthiamine-stripped serum (e.g., human serum). Another useful medium is apurely synthetic serum. Synthetic serums may consist solely of albumindissolved in water or saline or may contain other components found innormal human serum. Most importantly, the medium used for the blanks,positive controls, and/or calibration standards is substantially free ofthiamine (other than that added by the investigator). Preferably, themedium contains less than 6 nmol/L, and more preferably, less than 1-3nmol/L.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the invention.

EXAMPLE 1 Blood Sampling Initial Processing and Storage

Subjects were restricted from alcohol, coffee, tea, raw fish, rawshellfish, and vitamins consumption for 24 hours before blood samplecollection. An overnight fast was also required prior to blood samplecollection.

For plasma samples, blood was collected in light-protected tubescontaining EDTA or sodium heparin. Plasma was prepared within 4 hours ofsample collection by centrifugation of whole blood at 800-1000×g for8-10 minutes, at 2-8° C.

For serum samples, blood was collected in light-protected tubes andallowed to clot at about 2-8° C. for about 20-30 minutes, followed bycentrifugation at 800-1000×g for 8-10 minutes.

Immediately following collection and initial processing, serum, plasma,and whole blood samples were transferred to dark-brown polypropylene orpolyethylene transport tubes or to neutral color polypropylene orpolyethylene tubes that were wrapped in aluminum foil to protect thesamples from light. The samples were then frozen at −10 to −30° C. untilassay.

EXAMPLE 2 Sample Preparation for HPLC Analysis

An internal standard (IS) working solution of 1.0 μg/mL of pyrithiaminehydrobromide (Sigma-Aldrich, Catalog No. P0256) was prepared bydissolving 25 mg of pyrithiamine hydrobromide in 25 ml of 0.01N aqueousHCl and then diluting that solution 1:1000 with deionized water.

The samples (e.g., blood, serum, or plasma) were thawed, vortexed,aliquoted (400 μl of serum or plasma, 800 μl of whole blood), and spikedwith 100 μl (200 μl for whole blood sample) of the diluted pyrithiamineIS working solution.

External standard solutions of TPP were prepared. A 10 mM TPP stocksolution was prepared in 0.01 N aqueous HCl which was diluted 1:1000 indeionized water. This solution was used to prepare external standardsolutions of 350 nM, 175 nM, and 35 nM TPP in analyte-stripped,delipidized human serum (Biocell Labs; catalog no. 1131-00). A 400 μlaliquot of each external standard solution was spiked with 100 μl of ISworking solution and processed in parallel with the experimentalsamples. The external standard solutions were used for quality controland to ensure that the acid phosphatase reaction was performed tosubstantial completion.

Blanks (i.e., negative controls) containing no thiamine were alsoprepared and processed in parallel with the experimental samples. Blankscontained Biocell™ and IS.

All samples (i.e., experimental samples, external standards, and blanks)were subjected to a protein precipitation by adding 400 μl (800 μl forwhole blood samples) of 7% aqueous perchloric acid. The samples weremixed for about 5 minutes, centrifuged at 3500 rpm for 10 minutes at2-8° C. 400 μl of supernatant from each sample was recovered for furtherprocessing.

Next, the supernatants were mixed with an equal volume of 1.0 M sodiumacetate buffer (pH 4.6±0.1). 100 μl of acid phosphatase solution (10mg/ml in deionized water; Sigma-Aldrich, Catalog No. P3752) was added,mixed, and incubated at 40° C. for 1-2 hours (for plasma and serumsamples). Whole blood was incubated overnight.

The hydrolysis reaction was terminated by the addition of 2 mlchloroform, followed by a brief vigorous mixing, and centrifugation at3000 rpm for 10 minutes. 400 μl of the aqueous phase (top layer) wastransferred to a 96-well plate compatible with an autosampler injectorfor HPLC separation.

EXAMPLE 3 Sample Purification Using HPLC

The sample mixtures produced in Example 2 were subjected to HPLCpurification. Ten microliters of solution were injected onto CohesiveTLX LC system with a Luna C18(2), 50×2.0 mm, 5 μm HPLC column(Phenomenex, Catalog No. 00B-4252-B0)).

The elution from the HPLC column was driven by a binary step-gradient ofaqueous ammonium acetate and acetonitrile at a flow rate of 0.8mL/minute. The total duration for chromatograph is 240 seconds with adata acquisition window of 60 seconds. The mobile phase is altered asfollows:

% aqueous phase Time After Sample (10 mM % organic phase Injection (sec)ammonium acetate) (100% acetonitrile)  0 100% 0%  60 5% 95% 100 100% 0%240 End of run

The selected parameters for HPLC were as follows:

-   Injection volume: 10±4 μL-   Autosampler tray temperature: 5±2° C.-   Switch valve loop size: 200 μL

As shown in FIGS. 1 and 2, thiamine and the IS co-eluted at about 0.43minutes from an EDTA-plasma sample. The co-eluting thiamine and IS wasthen transferred to the MS/MS for quantification.

EXAMPLE 4 Sample Quantification Using MS/MS

Detection of thiamine and the IS was accomplished by electrospraytriplequad MS-MS system (Thermo Finnigan TSQ Quantum Ultra). The flow ofliquid solvent from the analytical column entered the heated nebulizerinterface of the MS/MS analyzer. The solvent/analyte mixture was firstconverted to vapor in the heated tubing of the interface. The analytes,contained in the nebulized solvent, were ionized. The ions passedthrough the orifice of the instrument and entered the first quadrapole.Quadrapoles 1 and 3 (Q1 and Q3) were the mass filters, allowingselection of ions based on their mass to charge ratio (m/z). Quadrapole2 (Q2) was the collision cell, where ions were fragmented by collisionwith argon molecules.

The first quadrapole of the MS/MS (Q1) selected for ionized thiaminewith an m/z of about 265.00 or the internal standard with an m/z ofabout 259.04 (“the parent ions”).

The parent ions passed to the collision chamber (Q2), while ions withany other m/z collided with the sides of the quadrapole (Q1) and weredestroyed. Ions entering Q2 were subjected to Collision-InducedDissociation (CID) by colliding with argon. The daughter ions generatedwere passed into quadrapole 3 (Q3) for detection. Two distinct daughterions were detected for thiamine: m/z=144.03 and m/z=121.94. A singledaughter ion was detected for the IS having m/z=122.00. Quantificationof sample thiamine is based on peak area ratio of two thiamine daughterions over the single IS daughter ion acquired by selective reactionmonitoring (SRM) in positive mode. The peak area ratio used forquantification was calculated by summing the peak areas of the twothiamine daughter ions (m/z 144.03 and 121.94) and dividing the resultby the peak area of the IS daughter ion (m/z 122.00).

Selected MS/MS parameters were:

-   Scan type: positive ion SRM-   Run time: 1.0±0.2 minutes-   MS/MS transitions: thiamine (265.00 parent ion to 144.03 and 121.94    daughter ions) internal standard (259.04 parent ion to 122.00    daughter ion)    MS common parameters:    -   spray voltage: 3400    -   sheath gas: 54    -   aux gas: 20    -   capillary temperature: 250° C.    -   tube lens offset: 101    -   collision energy: 10    -   scan width: 0.05    -   scan time: 0.1    -   Q1 resolution: 0.2    -   Q2 resolution: 0.7

EXAMPLE 5 Calibration and Standard Curves Using MS/MS

A stock solution of 10 mM thiamine in 0.01N aqueous HCl was prepared anddiluted to 10 μM in deionized water. Calibration standards of 6 nM, 15nM, 30 nM, 60 nM, 150 nM, 300 nM, and 600 nM, and a blank, each furthercontaining a consistent amount of IS, were prepared by further dilutionwith analyte-stripped, delipidized human serum (Biocell Labs; catalogno. 1131-00). The calibration standards were treated the same way as thesamples, as described above.

The calibration standards were used to generate a standard curve of peakarea ratio versus thiamine concentration. Peak area ratios for thiaminewere calculated summing the peak area of two thiamine daughter ions (m/z144.03 and 121.94) and dividing the result by the peak area of the ISdaughter ion (m/z 122.00). The standard curve was generated using linearregression with 1/x weighting for data reduction. An acceptablecalibration curve has a correlation coefficient (R²) of 0.9950 orbetter. An standard curve generated by this method is shown in FIG. 3.

For all experimental, external control, and blank samples, the peak arearatios were calculated and plotted against the standard curve toquantify the thiamine concentration present in the sample. Calculationsof the unknown concentration were performed using LCquan software inXcalibur (Thermo Finnigan).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the invention has beenspecifically disclosed by preferred embodiments and optional features,modification, improvement and variation of the inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this invention. The materials, methods, andexamples provided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

1. A method for determining the amount of total thiamine in a body fluidsample, comprising: (i) removing soluble protein from said sample; (ii)treating said sample with an acid phosphatase to convert phosphorylatedthiamine to thiamine; (iii) performing an organic solvent extraction ofsaid sample from step (i), wherein the result of said extraction is anorganic solvent phase and an aqueous phase; (iv) purifying said thiaminefrom said aqueous phase of step (iii) by liquid chromatography; and (v)determining the amount of thiamine by mass spectrometry, wherein theamount of total thiamine in said body fluid sample is determined.
 2. Themethod of claim 1, wherein said soluble protein is removed from saidbody fluid sample in step (i) by treating said sample with an acid. 3.The method of claim 1, wherein said liquid chromatography comprises highperformance liquid chromatography (HPLC).
 4. The method of claim 1,wherein step (v) comprises ionizing said thiamine to a parent ion havinga mass/charge ratio of 265.00±1.0.
 5. The method of claim 4, wherein theamount of said parent ion is determined.
 6. The method of claim 4,wherein said parent ion is fragmented into one or more daughter ions andwherein the amount of one or more of said daughter ions is determined.7. The method of claim 6, wherein said one or more daughter ionscomprises an ion having a mass/charge ratio of 144.00±1.0 or 121.94±1.0.8. The method of claim 4, wherein said thiamine is ionized byelectrospray ionization.
 9. The method of claim 6, wherein said parention is fragmented by collision-induced dissociation using an inert gas.10. The method of claim 1, wherein said body fluid sample is plasma,serum, or whole blood.
 11. The method of claim 1, wherein an internalstandard is added to said body fluid sample prior to step (i).
 12. Themethod of claim 11, wherein said internal standard is pyrithiamine. 13.The method of claim 12, wherein said pyrithiamine is ionized to a parention having a mass to charge ratio of 259.04±1.0.
 14. The method of claim13, wherein said pyrithiamine parent ion is fragmented into a daughterion having a mass to charge ratio of 122.00±1.0.
 15. A method fordetermining the amount of total thiamine in a body fluid sample,comprising: (i) treating said sample with an acid and removing theacid-insoluble protein; (ii) treating said sample with an acidphosphatase to convert phosphorylated thiamine to thiamine; (iii)performing an organic solvent extraction of said sample from step (ii),wherein the result of said extraction is an organic solvent phase and anaqueous phase; (iv) purifying said thiamine from said aqueous phase ofstep (ii) by high performance liquid chromatography (HPLC); (v) ionizingsaid purified thiamine from step (iii) into a parent ion; (vi) isolatingsaid parent ion by mass spectrometry; (vii) fragmenting said parent ioninto one or more daughter ions; and (viii) quantifying said one or moredaughter ions by mass spectrometry and relating the amount of said oneor more daughter ions to the concentration of total thiamine present insaid body fluid sample.
 16. The method of claim 15, wherein saidthiamine parent ion has a mass/charge ratio of 265.00±1.0.
 17. Themethod of claim 16, wherein said one or more daughter ions comprises anion having a mass/charge ratio of 144.00±1.0 or 121.94±1.0.
 18. Themethod of claim 15, wherein step (v) comprises electrospray ionizationand step (vii) comprises collision-induced dissociation using a inertgas.
 19. The method of claim 18, wherein the inert gas is argon.
 20. Themethod of claim 15, wherein an internal standard is added to said bodyfluid prior to step (i).
 21. The method of claim 20, wherein saidinternal standard is pyrithiamine.
 22. The method of claim 21, whereinsaid pyrithiamine is ionized to a parent ion having a mass to chargeratio of 259.04±1.0.
 23. The method of claim 22, wherein saidpyrithiamine parent ion is fragmented into a daughter ion having a massto charge ratio of 122.00±1.0.
 24. The method of claim 15, wherein saidacid in step (i) is 7% perchloric acid.
 25. The method of claim 15,wherein said organic solvent in step (iii) is chloroform.